The long range goals of this project are to elucidate the interactions between the known mediators of sperm capacitation (the alterations to sperm that occur obligatorily to sperm between mating and fertilization) and to describe the metaregulatory mechanisms that establish intracellular mediator concentrations and distributions. The more complete understanding of the mechanisms that control crucial cellular functions in sperm gained from these studies will allow more selective and effective intervention in the palliative sense required for so clinical infertility, and in the preventative sense required for sperm-directed antifertility measures. The specific aims of this project are: to delineate the mechanisms that function in maintaining resting intracellular [Ca2+] and pH in sperm (with particular attention to Na+/H+ exchange activity); to determine the regulatory roles of intracellular pH and free [Ca2+] as mediators of the initiation and hyperactivation of sperm motility; to define the role of phospholipid metabolism in the alteration of membrane permeability to Ca2+ that appears basic to hyperactivation of motility and the sperm acrosome reaction. Some powerful new tools are available for achievement of these goals. The fluorescent probes, carboxyfluorescein, quin-2 and fura-2, provide continuous, sensitive and specific means for determination and monitoring of cytosolic pH and free [Ca2+], respectively, after their generation in situ from permeant ester precursors. These probes will be used, together with monovalent and divalent cation ionophores, to systematically establish new equilibria of intracellular pH and free [Ca2+] while examining the consequences to sperm motility by video microscopy. These same probes, together with selective agonists and antagonists of ion transport, will also allow examination and characterization of mechanisms of cellular pH and [Ca2+] homeostasis and metaregulatory control - in experiments that systematically, but transiently, alter intracellular mediator concentrations. The role of phospholipid metabolism will be defined by an improved determination of lysophospholipid production during sperm capacitation with and without established pharmacological inducers. Phospholipase A2 will be purified with a new affinity chromatography technique and characterized with respect to control by known mediators. Phospholipase C of sperm membranes will be examined for possible metaregulatory coupling by GTP and Ca2+ to surface receptors.